Y Kido

Kanazawa University, Kanazawa-shi, Ishikawa-ken, Japan

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Publications (11)33 Total impact

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    ABSTRACT: Carnitine and acetylcarnitine are important for the acquisition of motility and maturation of spermatozoa in the epididymis. In this study, we examined the involvement of carnitine/organic cation transporter (OCTN) in carnitine and acetylcarnitine transport in epididymal spermatozoa of mice. Uptake of both compounds by epididymal spermatozoa was time-dependent and partially Na(+)-dependent. Kinetic analyses revealed the presence of a high-affinity transport system in the spermatozoa, with K(m) values of 23.6 and 6.57 muM for carnitine and acetylcarnitine respectively in the presence of Na(+). Expression of OCTN2 and OCTN3 in epididymal spermatozoa was confirmed by immunofluorescence analysis. The involvement of these two transporters in carnitine and acetylcarnitine transport was supported by a selective inhibition study. We conclude that both Na(+)-dependent and -independent carnitine transporters, OCTN2 and OCTN3, mediate the supply of carnitine and acetylcarnitine to epididymal spermatozoa in mice.
    Reproduction (Cambridge, England) 12/2007; 134(5):651-8. · 3.56 Impact Factor
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    ABSTRACT: To investigate the transport function of the blood-brain barrier (BBB), we employed an in vitro model of the BBB, consisting of a co-culture of porcine brain capillary endothelial cells (BCECs) with rat astrocytes. Porcine BCECs were cultured on a filter insert with rat astrocytes on the underlying plastic well. Rat astrocytes induced characteristic BBB properties of porcine BCECs, such as gamma-glutamyl-transpeptidase activity and intercellular adhesion of porcine BCECs. Next, the transport properties of P-glycoprotein (P-gp) substrate and several anionic compounds across the co-cultured porcine BCECs were characterized. Expression of P-gp was detected by immunocytochemistry, and efflux-directed transport of the P-gp substrate [(3)H]daunomycin was observed. Luminal-to-abluminal transport of the monocarboxylic acid transporter 1 (MCT1) substrate [(14)C]benzoic acid was saturable, and the K(m) value (3.05 mM) was similar to that for brain uptake observed in vivo. Abluminal-to-luminal transport of [(14)C]benzoic acid was also saturable, indicating that the monocarboxylic acid transporter of the BBB contributes to the efflux from the brain as well as to blood-to-brain influx. Abluminal-to-luminal transport of organic anions, [(3)H]dehydroepiandrosterone sulfate, [(3)H]estrone sulfate and [(3)H]estradiol 17beta-D-glucuronide was significantly higher than the corresponding luminal-to-abluminal transport. These results demonstrate the presence of multiple efflux transport pathways in this in vitro model.
    Drug Metabolism and Pharmacokinetics 02/2002; 17(1):34-41. · 2.07 Impact Factor
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    ABSTRACT: Transport of L-[3H]carnitine and acetyl-L-[3H]carnitine at the blood-brain barrier (BBB) was examined by using in vivo and in vitro models. In vivo brain uptake of acetyl-L-[3H]carnitine, determined by a rat brain perfusion technique, was decreased in the presence of unlabeled acetyl-L-carnitine and in the absence of sodium ions. Similar transport properties for L-[3H]carnitine and/or acetyl-L-[3H]carnitine were observed in primary cultured brain capillary endothelial cells (BCECs) of rat, mouse, human, porcine and bovine, and immortalized rat BCECs, RBEC1. Uptakes of L-[3H]carnitine and acetyl-L-[3H]carnitine by RBEC1 were sodium ion-dependent, saturable with K(m) values of 33.1 +/- 11.4 microM and 31.3 +/- 11.6 microM, respectively, and inhibited by carnitine analogs. These transport properties are consistent with those of carnitine transport by OCTN2. OCTN2 was confirmed to be expressed in rat and human BCECs by an RT-PCR method. Furthermore, the uptake of acetyl-L-[3H]carnitine by the BCECs of juvenile visceral steatosis (jvs) mouse, in which OCTN2 is functionally defective owing to a genetical missense mutation of one amino acid residue, was reduced. The brain distributions of L-[3H]carnitine and acetyl-L-[3H]carnitine in jvs mice were slightly lower than those of wild-type mice at 4 h after intravenous administration. These results suggest that OCTN2 is involved in transport of L-carnitine and acetyl-L-carnitine from the circulating blood to the brain across the BBB.
    Journal of Neurochemistry 01/2002; 79(5):959-69. · 3.97 Impact Factor
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    ABSTRACT: We present here the evidence of molecular and functional expression of LAT1 and LAT2, subunits of the large neutral amino acid transporter system L, in cultured brain capillary endothelial cells of the rat. By means of the RT-PCR method, transcripts of LAT1, LAT2 and heavy chain of 4F2 antigen (4F2hc) were detected in rat primary cultured brain capillary endothelial cells (BCECs) and immortalized subline, RBEC1. The uptake properties of RBEC1, such as [3H]leucine and L-[3H]DOPA uptake, were similar to those of primary cultured BCECs. So, RBEC1 may retain almost native properties of the large neutral amino acid uptake activities. [3H]Leucine uptake by RBEC1 showed two saturable components and the Km values of the high- and low-affinity components were 8.92+/-3.18 and 119+/-45 microM, respectively. The Km value of the high-affinity component agreed well with that of LAT1 and the amino acid transport selectivity of RBEC1 was similar to that of LAT1. Therefore, it is suggested that LAT1 is important at the blood-brain barrier of rats. Additionally, the Km value of the low-affinity component was similar to that of LAT2. These observations indicate that LAT1 and LAT2 are involved as transporters for large neutral amino acids at the blood-brain barrier. Additionally, we concluded that RBEC1 is useful as an in-vitro model for evaluation of the pharmacological relevance of system L at the blood-brain barrier.
    Journal of Pharmacy and Pharmacology 05/2001; 53(4):497-503. · 2.03 Impact Factor
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    ABSTRACT: Transport of new quinolone antibacterial agents (quinolones) at the blood-brain barrier (BBB) was studied in vitro by using immortalized rat brain capillary endothelial cells RBEC1, and in vivo by using the brain perfusion method in rats and multidrug-resistant mdr1a/1b gene-deficient mice. The permeability coefficient of grepafloxacin measured by brain perfusion was increased by an excess of unlabeled grepafloxacin, suggesting a participation of a saturable BBB efflux system. Uptake coefficients of [(14)C]grepafloxacin, [(14)C]sparfloxacin, and [(14)C]levofloxacin by RBEC1 cells at the steady state were increased in the presence of the unlabeled quinolones. The steady-state uptake of [(14)C]grepafloxacin was increased in the presence of various quinolones. Brain distributions of [(14)C]grepafloxacin and [(14)C]sparfloxacin evaluated in terms of the brain-to-plasma free concentration ratio in mdr1a/1b gene-deficient mice were significantly higher than those in wild-type mice, demonstrating an involvement of P-glycoprotein as the efflux transporter. Anionic compounds, including 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and genistein, increased the steady-state uptake of [(14)C]grepafloxacin by RBEC1 cells. Because [(14)C]grepafloxacin was transported by multidrug resistance-associated protein (MRP), in MRP1-overexpressing cells and because RBEC1 and primary cultured brain capillary endothelial cells expressed MRP1, this protein may be an additional efflux transporter for quinolones. Furthermore, the permeability coefficient of [(14)C]grepafloxacin across the BBB was increased by DIDS or in the absence of bicarbonate ions in the brain perfusion method. DIDS or bicarbonate ion did not affect MRP1 function. Accordingly, the brain distribution of quinolones is restricted by the action of multiple efflux transporters, including P-glycoprotein, MRP1, and an unknown anion exchange transporter.
    Journal of Pharmacology and Experimental Therapeutics 11/2000; 295(1):146-52. · 3.89 Impact Factor
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    ABSTRACT: The transport mechanism of the non-sedative H1-antagonist ebastine and its first-pass carboxylic acid metabolite carebastine at the blood-brain barrier (BBB) was studied. In rats, the brain uptake index (BUI) value of [14 C]carebastine was significantly lower than that of [14 C]ebastine. The BUI value of [14 C]carebastine was greatly increased by the addition of non-labeled carebastine. The steady-state uptake of [14 C]carebastine by P-glycoprotein-overexpressing K562/ADM cells was significantly lower than that by their parental drug-sensitive cell line K562. The decreased steady-state uptake of [14 C]carebastine by K562/ADM cells was reversed by verapamil. Steady-state uptake of [14 C]carebastine by primary cultured bovine brain capillary endothelial cells (bovine BCECs) was increased in the presence of metabolic inhibitors and verapamil. Non-labeled carebastine increased the steady-state uptake of a P-glycoprotein substrate, [3 H]vincristine, by bovine BCECs. The initial uptake of [3 H]mepyramine by bovine BCECs and RBEC1 (an immortalized cell line from rat brain capillary endothelial cells) was strongly inhibited by ebastine, while zwitterionic carebastine was slightly inhibitory. The values of brain-to-plasma unbound concentration ratio (Kp,f) in mdr1a(-/-) mice were increased 5.3-fold and 4.2-fold for [14 C ebastine and for [14 C]carebastine, respectively, compared with those in mdr1a(+/+) mice. Non-radiolabeled carebastine increased the Kp,f values of [14 C]carebastine in both types of mice. In conclusion, carebastine was shown to be a substrate for P-glycoprotein-mediated efflux from the brain at the BBB. A second efflux system may also be involved. The relatively low affinity of the uptake transport system for carebastine also limits the brain distribution of ebastine/carebastine.
    Journal of Drug Targeting 02/2000; 8(6):383-93. · 2.77 Impact Factor
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    ABSTRACT: To prove the functional significance of monocarboxylic acid transporter, MCT1 at the blood-brain barrier (BBB) for the passage of both endogenous and exogenous monocarboxylic acids into the central nervous system. Monocarboxylic acid transport at the BBB was studied in rats by using a newly established immortalized brain capillary endothelial cell (BCEC) line, RBEC1, and the results were compared with those obtained by using primary cultured BCECs, cells stably expressed with rat MCT1, and the in vivo brain uptake index (BUI) method. The cell line, RBEC1 meets various morphological and enzymatic criteria of BCECs and appears to be suitable for the study of BBB transport of monocarboxylic acids. The presence of MCT1-transcript in RBEC1 was confirmnned by the RT-PCR method, as previously observed in isolated brain capillaries. A typical substrate of MCT1, lactic acid, was taken up by RBEC1 in a stereospecific and saturable manner. The value of the kinetic parameter Km showed good agreement with values previously obtained in studies using an in vivo BUI and in vitro MCT1-transfected cells. An organic weak acid, benzoic acid, which has been considered to cross biological membranes by passive diffusion, exhibited carrier-mediated transport properties, such as saturation, pH dependence, and stereospecific inhibition in RBEC1, similar to those we observed in primary cultured rat BCECs. The Km values in RBEC1, in primary cultured BCECs and in the in vivo BUI method were comparable and well agreed with that obtained in MCT1-transfected cells, suggesting that the transport features of benzoic acid observed by in vitro methods well reflect the in vivo transport activity. Furthermore, hybrid depletion of MCT1 in RBEC1 using an antisense oligonucleotide against rat MCT1 abolished the saturable transport of benzoic acid. These observations show that MCT1 plays a significant role in the transport of monocarboxylic acids, including the exogenous organic weak acid benzoic acid, as well as native lactic acid.
    Pharmaceutical Research 02/2000; 17(1):55-62. · 4.74 Impact Factor
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    ABSTRACT: Monoclonal antibodies (MAbs) against kanamycin were prepared by using a kanamycin-bovine gamma-globulin conjugate for the immunization of mice. Splenocytes from BALB/c immunized mice were fused with P3X63Ag8U.1 myeloma cells. This resulted in two hybridoma cell lines. Fifty per cent inhibition concentrations (IC50) for the MAbs were 2 and 5 ng ml-1. One MAb (IC50 = 2 ng ml-1) was named #22 and was used to develop quantitative assays for kanamycin by means of an enzyme-linked immunosorbent assay (ELISA). The detection limit was 0.2 ng ml-1 and the standard deviations were 0.2-4.4% for intra-assay and 0.6-4.7% for inter-assay, respectively. The detection limits using peroxidase were 4 ppb in cattle milk, cattle plasma, cattle urine, swine plasma, swine urine and chicken plasma. Using the MAb #22 produced, a rapid test kit based on an immunochromatographic method was developed. The detection limits using the kit were 50 ppb in cattle milk, cattle plasma, cattle urine and chicken plasma.
    The Analyst 12/1999; 124(11):1611-5. · 3.97 Impact Factor
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    ABSTRACT: The participation of the monocarboxylic acid transporter MCT1 in the intestinal absorption of weak organic acids has been clarified by functional characterization, by use of stably transfected cells, and by immunohistochemical location of the transporter in intestinal tissues. Immunohistochemical analysis by use of the anti-MCT1 antibody showed that MCT1 is distributed throughout the upper and lower intestines, especially in the basolateral membrane and, to a lesser extent, in the brush-border membrane. When the transporter gene rat MCT1 was transfected into MDA-MB231 cells, transport of benzoic acid, a model weak organic acid that has been generally believed to be transported across the cell membranes by passive diffusion, and lactic acid in rat MCT1-transfected cells was significantly increased compared with transport in cells transfected with the expression vector pRc-CMV alone (mock cells). The observed transport was pH-dependent and activity increased between pH 7.5 and pH 5.5, whereas pH-dependence in mock cells was moderate. Rat MCT1-mediated benzoic acid uptake was saturable, with an apparent Km value of 3.05 mM. In addition, MCT1 increased the efflux of [14C]benzoic acid from the cells. Several weak organic acids were also transported by rat MCT1. These results show that pH-dependent intestinal absorption of weak organic acids, previously explained in terms of passive diffusion according to the pH-partition hypothesis, is at least partially accounted for by MCT1-mediated transport energized at acidic pH by utilization of the proton gradient as a driving force.
    Journal of Pharmacy and Pharmacology 11/1999; 51(10):1113-21. · 2.03 Impact Factor
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    ABSTRACT: The participation of the monocarboxylic acid transporter MCT1 in the intestinal absorption of weak organic acids has been clarified by functional characterization, by use of stably transfected cells, and by immunohistochemical location of the transporter in intestinal tissues.Immunohistochemical analysis by use of the anti-MCT1 antibody showed that MCT1 is distributed throughout the upper and lower intestines, especially in the basolateral membrane and, to a lesser extent, in the brush-border membrane. When the transporter gene rat MCT1 was transfected into MDA-MB231 cells, transport of benzoic acid, a model weak organic acid that has been generally believed to be transported across the cell membranes by passive diffusion, and lactic acid in rat MCT1-transfected cells was significantly increased compared with transport in cells transfected with the expression vector pRc-CMV alone (mock cells). The observed transport was pH-dependent and activity increased between pH 7.5 and pH 5.5, whereas pH-dependence in mock cells was moderate. Rat MCT1-mediated benzoic acid uptake was saturable, with an apparent Km value of 3.05 mM. In addition, MCT1 increased the efflux of [14C]benzoic acid from the cells. Several weak organic acids were also transported by rat MCT1.These results show that pH-dependent intestinal absorption of weak organic acids, previously explained in terms of passive diffusion according to the pH-partition hypothesis, is at least partially accounted for by MCT1-mediated transport energized at acidic pH by utilization of the proton gradient as a driving force.
    Journal of Pharmacy and Pharmacology. 09/1999; 51(10):1113 - 1121.
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    ABSTRACT: Monensin, a member of the ionophoric polyether antibiotics, is used primarily as a coccidiostat. A protein conjugate of monensin was prepared and utilized to produce monoclonal antibodies in the BALB/c-P3X63Ag8U.1 fusion system. Only one hybridoma that produces monoclonal antibody against monensin was isolated from one in 329 wells. The monoclonal antibody was used to develop quantitative assays for monensin by means of an enzyme-linked immunosorbent assay (ELISA). The detection limit was 1 ng ml-1 and the relative standard deviations were 2.1-6.3% intra-assay and 5.9-12.9% inter-assay. All ELISA results for assay of chicken plasma and cattle milk were confirmed using a bioassay to be used as the official method. The ELISA and bioassay results showed close correlations for plasma (r2 = 0.98, n = 25) and milk (r2 = 0.95, n = 25). Using the anti-monensin monoclonal antibodies produced, a rapid test kit based on the immunochromatographic method was developed. Detection limits of monensin for cattle milk, cattle plasma and chicken plasma were about 40, 40 and 160 ppb. respectively.
    The Analyst 01/1999; 123(12):2573-8. · 3.97 Impact Factor

Publication Stats

351 Citations
33.00 Total Impact Points

Institutions

  • 1999–2007
    • Kanazawa University
      • Graduate School of Natural Science and Technology
      Kanazawa-shi, Ishikawa-ken, Japan
    • Research Institute for Animal Science in Biochemistry & Toxicology
      Sagamihara, Kanagawa, Japan